Background correction,quantile normalization, and gene expression analysis were performed using RMA (Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003)A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics 19(2):185-193).
Performed with Agilent Genchip Scanner 2500 according to manufacturers instructions (Agilent Technologies UK Ltd., South Queensferry, West Lothian, EH30 9TG, United Kingdom)
(Parameters: Scanning hardware = OTHER: Agilent Genchip Scanner 2500, Scanning software = Scanning software)
Cells were harvested and total RNA extracted using the RNeasy kit (Qiagen).
(Parameters: Extracted product = total_RNA, Amplification = none)
Two strains, temperature sensitive pkc1ts (DL523) and wild-type PKC1 (DL100), were synchronised in S phase and then released for 60 minutes. DL100 (MATa leu20 trp1 ura30 his4 CAN1R) and DL523 (MATa ura30 trp1 his4 CAN1R pkc1::LEU2 Ycp50 Pkc1ts) were provided by David Levin. Yeast cultures were synchronised in S phase by hydroxyurea (HU) (200 mM) treatment for 2.5 hrs (YPD medium at 37 degrees C). At this point, time 0, HU samples for microarray were taken volume (5ml). HU was removed by spin down, wash and release in fresh media. Time 60 samples were taken after 60 minutes outgrowth (5 ml). The sensitivity of cells to hydroxyurea (200 mM) and 37oC was tested. Plates were incubated at either 30 degrees C or 37 degrees C for 3 days.